Organ-Specific Metabolome Deciphering Cell Pathways to Cope with Mercury in Wild Fish (Golden Grey Mullet Chelon auratus)

Metabolomics is a powerful approach in evaluating the health status of organisms in ecotoxicological studies. However, metabolomics data reflect metabolic variations that are attributable to factors intrinsic to the environment and organism, and it is thus crucial to accurately evaluate the metabolome of the tissue/organ examined when it is exposed to no stressor. The metabolomes of the liver and gills of wild golden grey mullet (Chelon auratus) from a reference area were analyzed and compared by proton nuclear magnetic resonance (1H NMR)-based metabolomics. Both organs were characterized by amino acids, carbohydrates, osmolytes, nucleosides and their derivatives, and miscellaneous metabolites. However, similarities and differences were revealed in their metabolite profile and related to organ-specific functions. Taurine was predominant in both organs due to its involvement in osmoregulation in gills, and detoxification and antioxidant protective processes in liver. Environmental exposure to mercury (Hg) triggered multiple and often differential metabolic alterations in fish organs. Disturbances in ion-osmoregulatory processes were highlighted in the gills, whereas differential impairments between fish organs were pointed out in energy-producing metabolic pathways, protein catabolism, membrane stabilization processes, and antioxidant defense system, reflecting the induction of organ-specific adaptive and defensive strategies. Overall, a strict correlation between metabolites and organ-specific functions of fish gills and liver were discerned in this study, as well as organ-specific cytotoxicity mechanisms of Hg in fish.

Pre-clinical validation of a novel pan-cancer CAR-T cell immunotherapy against nfP2X7

Chimeric antigen receptor (CAR)-T immunotherapy is a novel treatment that genetically modifies the patient’s own T cells to target and kill malignant cells. CAR-T cells demonstrated robust clinical activity against certain B-cell malignancies. However, identification of tumour-specific antigens expressed on multiple cancer types, especially on solid cancers, remains a major challenge. P2X purinoceptor 7 (P2X7) is an ATP gated cation channel that forms homotrimers and heterotrimers at the cell surface. When functioning normally, it controls ion transport in response to ATP. A dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from a range of tissues, while being undetectable on healthy cells. We generated prototype nfP2X7-targeting human CAR-T cells, which demonstrated effective antigen-specific cytotoxicity against twelve solid cancer types including breast, prostate, lung, colorectal, brain and skin in vitro. In preclinical xenograft mouse models of aggressive breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibited robust anti-tumour efficacy. These data indicate CAR-T cells targeting nfP2X7 have potential as a novel broad-spectrum cancer immunotherapy for solid tumours in humans.

The Diversity of Long-Term Persistent WT1-Specific Cytotoxic T Lymphocytes after Wilms' Tumor 1 Peptide Vaccination

[Introduction] Remarkable advances have been made in cancer immunotherapy, including the development of peptide-based cancer vaccines. Wilms' tumor 1 (WT1) is one of the cancer-testis antigens, and the WT1 gene is overexpressed in hematologic malignancies. Several clinical trials of WT1 peptide vaccines showed promising efficacy and high safety of the vaccines for hematologic malignancies. In these trials, immunological responses were assessed within 2 years after vaccination, and the transient WT1-specific immune response observed in many cases early after vaccination was confirmed. However, the long-term durability of the response of WT1-specific CD8+ cytotoxic T lymphocytes (CTLs) after peptide vaccine therapy and the T-cell receptor (TCR) diversity in those CTLs has not been clarified. More than 10 years ago, a patient with chronic myeloid leukemia (CML) received WT1 peptide vaccination after the failure of tyrosine kinase inhibitor therapy. After vaccination, WT1-specific CD8+ CTLs were observed. We continued the immunological assessment of the patient for more than 10 years after the WT1 peptide vaccination. Herein, we report our findings from the long-term monitoring of WT1-specific CTLs in the patient with CML and describe the results of our detailed analysis, including the functionality and clonality of the CTLs. [Methods] After obtaining written consent from a patient whose CML was difficult to control by imatinib, HLA-A*24:02-restricted modified-type WT1 peptide (WT1 peptide; 9 mer peptide of CYTWNGMNL) was administered to the patient. Post-vaccination, we followed up with the patient. Immune monitoring was performed using a WT1 tetramer assay after mixed lymphocyte peptide culture (MLPC assay). The limiting-dilution (mononuclear cells divided into 20 wells or more, equally containing 3 × 10 5 cells), 2-week cultures with WT1 peptide stimulation and counting of "positive wells" containing expanded WT1 tetramer+ CD8+ T cells were performed for the MLPC assay. The MLPC assay was used to detect functional WT1-specific CD8+ T cells that can expand in response to the WT1 peptide and estimate the frequency of these WT1-specific CD8+ T cells among all CD8+ T cells. For the functionality of WT1-specific CD8+ T cells, we evaluated WT1-specific cytotoxicity and cytokine production in the presence and absence of WT1 peptide pulse to T2A24 cells transfected with the GFP gene (T2A24-GFP). The phenotype and TCR of the WT1-specific CD8+ T cells expanded by MLPC were analyzed using flow cytometry and next-generation sequencing, respectively. [Results] After the WT1 peptide vaccination, the copy numbers of major bcr-abl transcripts gradually decreased, and a therapy free remission was achieved in the patient. No severe adverse effects were observed. The estimated frequency of WT1-specific CD8+ T cells peaked in the third year after vaccination (27 cells per 10 6 CD8+ T cells, 0.00027%) and then declined to 1 - 5 per 10 6 CD8+ T cells at 13 years after vaccination. The WT1-specific CD8+ T cells showed that WT1 peptide-specific cytotoxicity and WT1 peptide-specific IFN-γ release in vitro. These WT1-specific CTLs had different TCRs in each MLPC well. This result was confirmed by three independent analyses, and no common TCRs were detected. Twelve different TCRs were detected in the three analyses. [Conclusion] The WT1 peptide vaccine successfully generated long-lasting and diverse WT1-specific immune responses in a patient with CML. The WT1 peptide vaccine may be a efficient immune therapy for CML patients. Disclosures No relevant conflicts of interest to declare.

STEM-12. THE SMALL MOLECULE DRUG CBL0137 INCREASES THE LEVEL OF DNA DAMAGE AND THE EFFICACY OF RADIOTHERAPY FOR GLIOBLASTOMA

Glioblastoma (GBM) is a fatal and incurable brain tumor, with an average life expectancy after diagnosis of only 12-15 months. A main reason for the lethality of GBM is inevitable recurrence, caused by a small population of the tumor cells, called cancer stem cells (CSCs). These cells are aggressive, infiltrative, and resistant to current GBM treatments of chemotherapy and radiotherapy. We use a small molecule drug, CBL0137, which inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy and hence lead to increased CSC death and prolonged survival in preclinical models. Clonogenic assays were used to show that CSCs were radiosensitized after CBL0137 treatment. We saw increased DNA damage when GBM CSCs were treated with CBL0137, as well as a decrease in foci resolution over time, when CBL0137 was combined with irradiation. In order to elucidate if the increase in DNA damage was directly due to the inhibition of the FACT complex, we depleted the level of FACT in our GBM CSCs. FACT depletion also led to increased DNA damage, and even more so when combined with irradiation. To validate whether combination therapy sensitized CSCs to radiotherapy in vivo, we used a subcutaneous mouse model and showed combination treatment decreased CSCs frequency in these tumors as well as decreased tumor volume. With an orthotopic model of GBM, we showed that CBL0137 treatment followed by radiotherapy significantly increased survival of mice bearing tumors over either treatment alone. Together, this work establishes a new treatment paradigm for GBM, which sensitizes radio-resistant GBM CSCs to irradiation, a critical component of patient care. Radio-sensitizing agents, including CBL0137, pose an exciting new therapeutic capable of increasing the efficacy of irradiation, by inclusively targeting CSCs.

STEM-22. TARGETING REPLICATION STRESS RESPONSE FOR GLIOMA STEM CELL SPECIFIC CYTOTOXICITY

BACKGROUND Evidence suggests treatment resistant glioma stem cells (GSCs) drive glioblastoma (GBM) recurrence. Current treatments fail to eradicate GSC and novel GSC targeting therapies are a priority. GSC exhibit elevated DNA replication stress (RS) versus non GSC tumour cells driving constitutive DNA damage response (DDR) activation and efficient DNA repair. We previously demonstrated that targeting RS response with combined ATR and PARP inhibition (CAiPi) (VE821 and Olaparib) provides potent GSC specific cytotoxicity. In this study we investigated the underlying DDR phenotype which determines this vulnerability. RESULTS Paired GSC enriched (‘GSC’) and GSC depleted, differentiated (‘bulk’) populations were cultured from GBM specimens in neurobasal media with growth factors or serum containing media respectively. GSC exhibited reduced survival following exposure to CAiPi versus bulk. CAiPi significantly increased 53BP1 G1 phase nuclear bodies (53BP1NBs) in GSC, which are known to shield under-replicated DNA in actively transcribed genes. Mapping the genomic distribution of endogenously occurring replication dependent DNA double strand breaks via Breaks Ligation In Situ Sequencing (BLISS), revealed reduced intragenic DSB in long actively transcribed genes in GSC versus bulk at baseline, suggesting a reliance upon transcription coupled DSB repair in GSC. RNA seq demonstrated CAiPi-induced transcriptomic alterations in GSC including replication regulation and initiation. DNA fibre assay showed that CAiPi increased GSC new origin firing which correlated with PARP trapping. GSCs were rescued from CAiPi by roscovitine induced inhibition of excess origin firing. CAiPi is potently radiosensitizing by clonogenic assay and we demonstrated murine blood brain barrier penetration of CAiPi utilising VE822 and pamiparib in vivo. CONCLUSION Dysregulation of origin firing by CAiPi exposes a GSC specific vulnerability which results in DNA under-replication and abrogation of proficient DNA repair seen at long actively transcribed genes and has potential to be clinically translated as a GSC specific cytotoxic therapy.

Air plasma-activated medium exerts tumor-specific cytotoxicity via the oxidative stress-induced perinuclear mitochondrial clustering

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy eliminating cancers effectively and safely is the current clinical choice. Since aggressive tumors have inherent or acquired resistance to multidisciplinary therapies targeting apoptosis, tumor-specific induction of another cell death modality is a promising avenue to meet the goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can induce cell death in OS and OC via a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) because of the characteristic unipolar mitochondrial perinuclear aggregation. APAM had potent antitumor activity both in vitro and in vivo. APAM caused apoptosis, necrotic cell death, and autophagy. APAM contained hydrogen peroxide and increased mitochondrial ROS (mROS), while the antioxidant N-acetylcysteine (NAC) prevented cell death. MPMC occurred following mitochondrial fragmentation coinciding with nuclear damages. MPMC was accompanied by the tubulin network remodeling and mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC and the microtubule inhibitor, Nocodazole. Increased Cardiolipin (CL) oxidation was also seen, and NAC and the peroxy radical scavenger Ferrostatin-1 prevented it. In contrast, in fibroblasts, APAM induced minimal cell death, mROS generation, mLPO accumulation, CL oxidation, and MPMC. These results suggest that MPMC is a tumor-specific cause of cell death via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC might serve as a promising target for exerting tumor-specific cytotoxicity.

Cytotoxic CD4+ T cells driven by T-cell intrinsic IL-18R/MyD88 signaling predominantly infiltrate Trypanosoma cruzi-infected hearts

Increasing attention has been directed to cytotoxic CD4+ T cells (CD4CTLs) in different pathologies, both in humans and mice. The impact of CD4CTLs in immunity and the mechanisms controlling their generation, however, remain poorly understood. Here, for the first time, we showed that CD4CTLs abundantly differentiate during mouse infection with an intracellular parasite. CD4CTLs appear in the spleen in parallel to Th1 cells, display pathogen-derived peptide-specific cytotoxicity against antigen-presenting cells and express immunoregulatory and/or exhaustion markers. We demonstrated that CD4CTL absolute numbers and activity are severely reduced in both Myd88-/- and Il18ra-/- mice. Of note, the infection of mixed-bone marrow chimeras revealed that WT, but not Myd88-/-, cells transcribe the CD4CTL gene signature and that Il18ra-/-CD4+ phenocopy Myd88-/-CD4+ T cells. Moreover, the adoptive transfer of WT CD4+GzB+ T cells to susceptible Il18ra-/- mice increased their survival. Importantly, cells expressing the CD4CTL phenotype predominate among CD4+ T cells infiltrating the infected cardiac tissue, are increased in the circulation of Chagas patients and their frequency correlates with severe cardiomyopathy. Our findings describe CD4CTLs as a major player in immune response to a relevant human pathogen and disclose T-cell intrinsic IL-18R/MyD88 signaling as a key pathway controlling the magnitude of the CD4CTL response.

Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in B cell malignancies. However, high tumor burden limits clinical efficacy and increases the risk of cytokine release syndrome and neurotoxicity, which is associated with over-activation of the CAR-T cells. The hinge domain plays an important role in the function of CAR-T cells. We hypothesized that deletion of glycine, an amino acid with good flexibility, may reduce the flexibility of the hinge region, thereby mitigating CAR-T cell over-activation. This study involved generating a novel CAR by deletion of two consecutive glycine residues in the CD8 hinge domain of second-generation (2nd) CAR, thereafter named 2nd-GG CAR. The 2nd-GG CAR-T cells showed similar efficacy of CAR expression but lower hinge flexibility, and its protein affinity to CD19 protein was lower than that of 2nd CAR-T cells. Compared to the 2nd CAR-T cells, 2nd-GG CAR-T cells reduced proinflammatory cytokine secretion without diminishing the specific cytotoxicity toward tumor cells in vitro. Furthermore, 2nd-GG CAR-T cells prolonged overall survival in an immunodeficient mouse model bearing NALM-6 when tumor burden was high. This study demonstrated that a lower-flexibility of CD8α hinge improved survival under high tumor burden and reduced proinflammatory cytokines in preclinical studies. While there is potential for improved safety and efficacy, yet this needs validation with clinical trials.

Lactose-conjugated porphyrin: Synthesis and photobiological evaluation as potential agents for photodynamic therapy

Porphyrin-based photosensitizers are conventional photodynamic agents applied in clinic. However, their clinic application has been overshadowed by the poor water solubility. In addition, they have weak tumor selectivity, which may cause undesirable side effects. The preparation of novel porphyrin derivatives has been explored for the potential application in photodynamic therapy (PDT). To achieve this goal, lactose-conjugated porphyrin nanoparticles (Lac-PorNPs) has been synthesized and characterized. PDT with Lac-PorNPs exhibits tumor-specific cytotoxicity in lactose receptor overexpressed HepG2 cells in vitro and in vivo. In summary, we designed and synthesized lactose conjugates porphyrin with enhanced water-solubility and tumor selectivity. This work expanded the application range of porphyrin-based photosensitizers for cancer treatment.